a. Streptomyces clavuligerus has been described in detail by Higgens et al, Int.J.Systematic Bacteriology, 21, 326 (1971). This streptomycete was of interest because it produced certain .beta.-lactam antibiotics such as penicillin N, 7-(5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid and 7-(5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4- carboxylic acid. The streptomycete has been deposited in the Agricultural Research Service Collection as NRRL 3585 and in the American Type Culture Collection as ATCC 27064. Streptomyces clavuligerus has also been referred to in U.S. Pat. No. 3,770,590 and also by Nagarajan et al, J.Amer.Chem.Soc., 93, 2308 (1971), Brannon et al, Antimicrob. Agents Chemother., 1, 237 (1972) and Antimicrob. Agents Chemother, 1, 247 (1972) and Higgens et al, J.Antibiotics, 27, 298 (1974).
b. .beta.-lactamases are enzymes which open the .beta.-lactam ring of penicillins and cephalosporins to give products which are devoid of antibacterial activity. These enzymes are produced by many bacteria, notably species or strains of Escherichia, Klebsiella, Proteus, Pseudomonas, Enterobacter and Staphylococcus and are in many instances the explanation for the resistance of certain strains of such organisms to some penicillins and cephalosporins. The importance of .beta.-lactamase production may be understood when it is realised that a high proportion of clinically isolated organisms produce .beta.-lactamases (see, for example, M. Wilson and I. A. Freeman, Bacteriological Proceedings, 80 (1969) where in a paper entitled `Penicillin Inactivation by Gram-negative Bacilli` they showed that 84% of the gram-negative organisms isolated in an American hospital produced .beta.-lactamase. In many cases, some penicillins or cephalosporins are ineffective in treating diseases ascribed to non .beta.-lactamase-producing organisms because of the common occurrence of co-infection by a .beta.-lactamase producer (see, for example, R. May et al; Brit. J.Dis.Chest., 66, 185 (1972). Combination of a .beta.-lactamase inhibiting substance with a penicillin or cephalosporin might be expected to protect the latter from degradation by bacterial .beta.-lactamase and thereby enhance their antibacterial activity against many infective organisms. This process of enhancement of the antibacterial activity is called synergism when the antibacterial activity of the combination is well in excess of the simple addition of the activities of the two separate substances. The .beta.-lactamase inhibiting component of the mixture is referred to as a synergist and such substances are valuable for increasing the antibacterial activity of penicillins and cephalosporins against resistant organisms. It is one of the objects of this invention to provide such synergists.
c. Examples of the use of certain .beta.-lactamase resistant semi-synthetic penicillins and cephalosporins as .beta.-lactamase inhibitors and synergists for penicillins and cephalosporins have already been described in the literature, see for example, Sutherland et al., Nature, 201, 868 (1964); Sabath et al., Nature, 204, 1066 (1964); O'Callaghan et al., Antimicrob. Agents and Chemotherapy, 1968, 67 (1969). However, none of these known agents have a dramatic effect on the spectrum of the other antibiotic present in the mixture.
d. Certain actinomycete cultures have been described as producing .beta.-lactamase inhibiting substances which act synergistically with penicillins or cephalosporins, for example, those cultures disclosed in British Pat. No. 1,363,075 and those described by Hata et al, J. Antibiotics, 25, 473 (1972) and Umezawa et al, J. Antibiotics, 26, 51 (1973). None of these .beta.-lactamase inhibitors of actinomycetal origin have yet been found to be of use in the clinic. Particularly noteworthy features which distinguish clavulanic acid from other .beta.-lactamase inhibitors of actinomycetal origin are its extractability into organic solvents from culture filtrate at pH2, its high stability in human blood and its broad spectrum of anti-bacterial and .beta.-lactamase inhibiting activity, its low molecular weight and its high R.sub.f values on paper chromatography using a variety of solvent systems.